College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, P.R. China.
College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, P.R. China.
J Dairy Sci. 2020 Dec;103(12):12091-12103. doi: 10.3168/jds.2020-18767. Epub 2020 Oct 1.
The major circadian clock gene PER2 is closely related to cell proliferation and lipid metabolism in various nonruminant cell types. Objectives of the study were to evaluate circadian clock-related mRNA abundance in cultured goat ruminal epithelial cells (REC), and to determine effects of PER2 on cell proliferation and mRNA abundance of short-chain fatty acid (SCFA) transporters, genes associated with lipid metabolism, cell proliferation, and apoptosis. Ruminal epithelial cells were isolated from weaned Boer goats (n = 3; 2 mo old; ∼10 kg of body weight) by serial trypsin digestion and cultured at 37°C for 24 h. Abundance of CLOCK and PER2 proteins in cells was determined by immunofluorescence. The role of PER2 was assessed through the use of a knockout model with short interfering RNA, and sodium butyrate (15 mM) was used to assess the effect of upregulating PER2. Both CLOCK and PER2 were expressed in REC in vitro. Sodium butyrate stimulation increased mRNA and protein abundance of PER2 and PER3. Furthermore, PER2 gene silencing enhanced cell proliferation and reduced cellular apoptosis in isolated REC. In contrast, PER2 overexpression in response to sodium butyrate led to lower cellular proliferation and ratio of cells in the S phase along with greater ratio of cells in the G/M phase. Those responses were accompanied by downregulated mRNA abundance of CCND1, CCNB1, CDK1, and CDK2. Among the SCFA transporters, PER2 silencing upregulated mRNA abundance of MCT1 and MCT4. However, it downregulated mRNA abundance of PPARA and PPARG. Overexpression of PER2 resulted in lower mRNA abundance of MCT1 and MCT4, and greater PPARA abundance. Overall, data suggest that CLOCK and PER2 might play a role in the control of cell proliferation, SCFA, and lipid metabolism. Further studies should be conducted to evaluate potential mechanistic relationships between circadian clock and SCFA absorption in vivo.
主要的生物钟基因 PER2 与各种非反刍动物细胞类型的细胞增殖和脂质代谢密切相关。本研究的目的是评估培养的山羊瘤胃上皮细胞（REC）中与生物钟相关的 mRNA 丰度，并确定 PER2 对细胞增殖和短链脂肪酸（SCFA）转运体、与脂质代谢、细胞增殖和凋亡相关的基因的 mRNA 丰度的影响。通过连续胰蛋白酶消化从断奶的布尔山羊（n = 3；2 月龄；~10 kg 体重）中分离瘤胃上皮细胞，并在 37°C 下培养 24 小时。通过免疫荧光测定细胞中 CLOCK 和 PER2 蛋白的丰度。通过使用短干扰 RNA 的敲除模型评估 PER2 的作用，并使用 15 mM 丁酸钠评估上调 PER2 的作用。体外 REC 中均表达 CLOCK 和 PER2。丁酸钠刺激增加了 PER2 和 PER3 的 mRNA 和蛋白丰度。此外，PER2 基因沉默增强了分离的 REC 中的细胞增殖并减少了细胞凋亡。相反，丁酸钠诱导的 PER2 过表达导致细胞增殖减少和 S 期细胞比例降低，同时 G/M 期细胞比例增加。这些反应伴随着 CCND1、CCNB1、CDK1 和 CDK2 的 mRNA 丰度降低。在 SCFA 转运体中，PER2 沉默上调了 MCT1 和 MCT4 的 mRNA 丰度。然而，它降低了 PPARA 和 PPARG 的 mRNA 丰度。PER2 的过表达导致 MCT1 和 MCT4 的 mRNA 丰度降低，而 PPARA 的丰度增加。总体而言，数据表明 CLOCK 和 PER2 可能在控制细胞增殖、SCFA 和脂质代谢中发挥作用。应进一步开展研究，以评估体内生物钟和 SCFA 吸收之间的潜在机制关系。
